Loss of the Pro-Apoptotic BH3-only Bcl-2 Family Member Bim Inhibits BCR Stimulation–induced Apoptosis and Deletion of Autoreactive B Cells

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion

Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics

Background: The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.Methods and Findings: We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.Conclusions: Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy

Chemical chaperone TUDCA prevents apoptosis and improves survival during polymicrobial sepsis in mice

Sepsis-induced lymphopenia is a major cause of morbidities in intensive care units and in populations with chronic conditions such as renal failure, diabetes, HIV and alcohol abuse. Currently, other than supportive care and antibiotics, there are no treatments for this condition. We developed an in vitro assay to understand the role of the ER-stress-mediated apoptosis process in lymphocyte death during polymicrobial sepsis, which was reproducible in in vivo mouse models. Modulating ER stress using chemical chaperones significantly reduced the induction of the pro-apoptotic protein Bim both in vitro and in mice. Furthermore, in a ‘two-hit’ pneumonia model in mice, we have been able to demonstrate that administration of the chemical chaperone TUDCA helped to maintain lymphocyte homeostasis by significantly reducing lymphocyte apoptosis and this correlated with four-fold improvement in survival. Our results demonstrate a novel therapeutic opportunity for treating sepsis-induced lymphopenia in humans

A novel BH3 ligand that selectively targets Mcl-1 reveals that apoptosis can proceed without Mcl-1 degradation

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemoresistance and disease relapse. However, unlike other prosurvival Bcl-2–like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)–only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, BimS2A, which is highly selective for Mcl-1. Unlike Noxa, BimS2A is unable to trigger Mcl-1 degradation, yet, like Noxa, BimS2A promotes cell killing only when Bcl-xL is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Cloning and growth-regulated expression of the gene encoding the hepatitis B virus middle surface antigen in Yarrowia lipolytica

Expression of the gene encoding the hepatitis B virus middle surface antigen (pre-HBsAg) in the yeast Yarrowia lipolytica has been studied. The preS2-HBsAg gene was expressed from the alkaline extracellular protease-encoding gene (XPR2) promoter. In the fusion construct, the membrane-spanning 'a' domain of preS2-HBsAg has been replaced by the leader peptide and the prol region of the alkaline protease, thus eliminating the epitope responsible for the immune escape mechanism. Expression has been found to be growth-stage dependent with the highest protein accumulation during the stationary phase, accounting for around 2.35% of the total soluble intracellular proteins. The produced protein was assembled into Dane particles and was immunogenic in mice. The expression vector was found to be stable for at least 100 generations under non-selective conditions

Trim33 Binds and Silences a Class of Young Endogenous Retroviruses in the Mouse Testis; a Novel Component of the Arms Race between Retrotransposons and the Host Genome

Transposable elements (TEs) have been active in the mammalian genome for millions of years and the silencing of these elements in the germline is important for the survival of the host. Mice carrying reporter transgenes can be used to model transcriptional silencing. A mutagenesis screen for modifiers of epigenetic gene silencing produced a line with a mutation in Trim33; the mutants displayed increased expression of the reporter transgene. ChIP-seq of Trim33 in testis revealed 9,109 peaks, mostly at promoters. This is the first report of ChIP-seq for Trim33 in any tissue. Comparison with ENCODE datasets showed that regions of high read density for Trim33 had high read density for histone marks associated with transcriptional activity and mapping to TE consensus sequences revealed Trim33 enrichment at RLTR10B, the LTR of one of the youngest retrotransposons in the mouse genome, MMERVK10C. We identified consensus sequences from the 266 regions at which Trim33 ChIP-seq peaks overlapped RLTR10B elements and found a match to the A-Myb DNA-binding site. We found that TRIM33 has E3 ubiquitin ligase activity for A-MYB and regulates its abundance. RNA-seq revealed that mice haploinsufficient for Trim33 had altered expression of a small group of genes in the testis and the gene with the most significant increase was found to be transcribed from an upstream RLTR10B. These studies provide the first evidence that A-Myb has a role in the actions of Trim33 and suggest a role for both A-Myb and Trim33 in the arms race between the transposon and the host. This the first report of any factor specifically regulating RLTR10B and adds to the current literature on the silencing of MMERVK10C retrotransposons. This is also the first report that A-Myb has a role in the transcription of any retrotransposon